The human immunodeficiency virus-I (HIV-I, also LAV, HTLV-III, or ARV) is the primary etiologic agent of the acquired immune deficiency syndrome (AIDS) [Barre-Sinoussi et al., Science, 220:868-871 (1983); Gallo et al., Science 224:500-503 (1984); Levy et al., Science 225:840-842 (1984)]. In addition to the gag, pol and env genes that encode the major virus structural proteins, the genome of HIV contains several other open reading frames designated sor, tat, art/trs and 3'-orf which encode additional viral proteins. These proteins are known to serve important regulatory functions during the HIV infectious cycle.
As used herein, the term "gene" connotes the DNA sequence information that specifies the expression of a particular protein product.
As first described by Haseltine and Wong-Staal and their colleagues, one important regulatory protein, the product of the tat gene, mediates the activation of the expression of gene sequences linked to the HIV LTR promoter region [Sodroski et al., Science 227:272-173 (1985); Arya et al., Science 229:69-73 (1985); Sodrocski et al., Science 229:74-77 (1985)]. This positive control of gene expression by the tat protein is an example of a phenomenon termed trans-activation. Trans-activation refers to the positive regulation of the expression of a specific target gene by a specific regulatory protein.
Positive-acting regulatory proteins and corresponding responsive target genes are well known in both prokaryotic and eukaryotic cell systems, as well as in many viruses. For example, many other animal viruses, as in the case of HIV, encode trans-activator proteins that are known to positively regulate viral gene expression. These viruses includes herpes viruses (such as herpes simplex virus, cytomegalovirus), adenoviruses, papovaviruses (such as SV40, polyoma virus, JC virus), leukemia retroviruses (such as bovine leukemia virus, HTLV-I, HTLV-II) and other lentiviruses (such as visna virus, equine infectious anemia virus, simian immunodeficiency virus). In addition to positive-acting regulatory proteins, examples of endogenous cellular and viral negative-acting regulatory proteins are known that can negatively regulate, i.e., repress, the expression of specific target genes.
The sequence information within the HIV LTR that is responsive to trans-activation by tat residues between nucleotides -17 and +58 (numbering is relative to the transcription start site) [Rosen et al., Cell 41:813-823 (1985); Muesing et al., Cell 48:691-701 (1987)]. This trans-activation target sequence may be directly recognized by the tat protein. Activation of expression of LTR-linked sequence by tat involves can increase in mRNA accumulation [Cullen, Cell 46:973-982 (1986); Gendelman et al., Proc. Natl. Acad. Sci. 83:9759-9763 (1986); Wright et al., Science 234: 988-934 (1986); Peterlin et al., Proc. Natl. Acad. Sci. 83:9734-9738 (1986); Musing et al., Cell 48:691-701 (1987)] and possibly post-transcription regulation [Rosen et al., Nature 319:555-559 (1986); Cullen, Cell 46:973-982 (1986); Feinberg et al., Cell 46:807-817 (1986)].
The derivation of stable human cell lines containing integrated heterologous genes expressing functional HIV tat [Rosen et al., J. Virol. 57:379-384 (1986); Cullen, Cell 46:973-982 (1986)] or a gene containing the chloramphenicol acetyl transferase (CAT) coding sequence linked to a HIV LTR [Wright et al., Science 234:988-992 (1986)] has been previously reported. Wright et al. showed further that the integrated LTR-cat gene was inducible by a transiently introduced tat-expressing gene. However, no previous report describes the derivation of stable human cell lines containing both the tat-expressing gene and the LTR-linked indicator gene.
The tat gene product is known to be essential for HIV replication [Dayton et al., Cell 44:941-947 (1986); Fisher et al., Nature 320:367-371 (1986)]. It is of considerable interest to determine the molecular mechanism by which the tat gene product induces gene expression. Since tat is an essential and virus-specific component of the HIV life cycle, specific inhibitors of tat function are likely to be important anti-HIV viral therapeutic agents. The present invention provides a system that allows for rapid screening and identification of compounds that specifically interfere with HIV tat function. The ability to identify compounds that selectively attack a virus without harming its mammalian host, offers the potential for effective yet nontoxic anti-viral drugs.
Importantly, the system we describe for the identification of inhibitors of HIV lends itself to simple, semi-automated nonradioactive assays that are free of infectious HIV. Moreover, the experimental strategy and screening methodology demonstrated here for HIV could be applied to other viral and mammalian cell systems for the identification of inhibitors of other specific positive-acting or negative-acting regulators of gene expression. Thus, the system demonstrated here is not limited to the identification of anti-viral therapeutic agents and could be applied to the identification of therapeutic agents for other disease conditions.